Type of lettuce and methods of production

ABSTRACT

The present invention relates to an iceberg lettuce having one or more romaine lettuce characteristics. The invention further relates to methods for producing iceberg lettuce varieties containing one or more romaine lettuce characteristics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/336,422, filed on Dec. 23, 2011, which is a continuationapplication of U.S. application Ser. No. 12/348,882, filed on Jan. 5,2009, now U.S. Pat. No. 8,101,817, which is a continuation of Ser. No.10/665,029, filed on Sep. 16, 2003, now U.S. Pat. No. 7,563,952, whichis a divisional of U.S. patent application Ser. No. 09/822,817, filedMar. 30, 2001, now U.S. Pat. No. 6,689,941, all of which areincorporated in their entirety herein by reference.

FIELD OF INVENTION

The present invention relates to the field of plant breeding. Moreparticularly, the present invention describes a lettuce plant typehaving the general shape of a romaine lettuce and having other qualitiessimilar to iceberg lettuce. This new iceberg lettuce plant type has anelliptical plant shape, and spatulate shaped leaves generallyrecognizable in stature and appearance similar to romaine lettuce,instead of the normal spherical shaped plants with obovate shaped leavesof the iceberg. Other qualities of these plants, e.g., head weight,interior and exterior color, texture, density, and taste, are similar toiceberg lettuce.

BACKGROUND OF THE INVENTION

Lettuce, Lactuca sativa L., is a commercially important fresh leaf cropbelonging to the Cichoreae tribe of the aster family (Asteraceae) whichincludes such other important crops as sunflowers and artichokes.Lettuce is widely grown throughout the temperate and subtropical regionsof the world and is used predominantly as a fresh green in the humandiet.

There are six morphological types of lettuce: iceberg (crisphead),romaine (cos), butterhead, leaf, stem and Latin. Ryder, E. J., 1979,Leafy Salad Vegetables, AVI Publishing Company. These basic lettucetypes frequently form the basis for grouping lettuces as is commonlyseen in supermarkets, grocery and produce stores.

The crisphead type is the most common in the United States, whilebutterheads and romaines are the popular types in northern and southernEurope. Id. In the United States, California is the leading producer oflettuce. California produces crisphead, leaf and romaine lettuce. In1995, California's cash receipts for crisphead lettuce amounted to about$987 million dollars. 1996 California Agricultural Resource Directory.Furthermore, also in 1995, California exported about $154 milliondollars of crisphead lettuce to other countries such as Japan, Canada,the European Union and Korea. Id.

Terms used herein to describe plants are explained in “Guidelines forthe Conduct of Tests for Distinctness, Homogeneity and Stability” UPOVRef. No. TG/13/7, 16 October 1993, which is hereby incorporated byreference.

The iceberg group lettuces are characterized by their relatively large,spherical, dense heads averaging 20.0 cm in diameter and 1000 g inweight, which are borne on a set of frame leaves that form the base ofthe plant. The heads are composed of leaves that are spirally arrangedon a stem with greatly foreshortened internodes, are tightly claspingupon one another forming a sphere of broadly shaped (obovate) leaves,where the length and width of each leaf is nearly identical, havinglength to width ratio (L/W ratio) of 1.0 approximates 20.0 cm in lengthto 20.0 in width. The length to width ratio for commercial iceberglettuce varieties ranges from about 0.5 to 1.0. Outer leaves range incolor intensity from dark green (Royal Horticultural Society Color Chart146A) to green (RHS 146B) with inner leaves ranging from very pale green(RHS 145C) to white or blanched (RHS 145D). Iceberg lettuces have aclosed head formation. Iceberg leaves have a high content of water—hencethe name, “crisphead.” More information regarding the generalcharacteristics of iceberg lettuce may be found in Ryder, E. J., LeafySalad Vegetables, AVI Publishing Company, which is hereby incorporatedby reference.

The romaine group of lettuces are characterized by large, cylindrical,semi-firm heads averaging 30.0 cm in diameter and 800 g in weight, whichare borne on a set of frame leaves that form the base of the plant. Theheads are composed of leaves that are spirally arranged on a stem withgreatly foreshortened internodes, are loosely clasping upon one anotherforming a roll of elongated, spatula-shaped (spatulate) leaves, wherethe length is normally 50% longer than the width, having a range oflength to width ratios of 1.2 to 2.5, where 1.5 is most common. Romainelettuces generally have a semi-open head formation. The name “romaine”comes from the French for “Roman”. Outer leaves range in color intensityfrom dark green (RHS 146A) to mid-green (RHS 146B) to light green (RHS146C) with inner leaves ranging from green (RHS 146B) to light green(RHS 146D). More information regarding the general characteristics ofromaine lettuce may be found in Ryder, E. J., Leafy Salad Vegetables,AVI Publishing Company.

A major disadvantage of existing iceberg varieties lies in their shape.While they are widely used in salads and sandwiches for their uniquetaste and crunchy texture, the iceberg's round shape makes it difficultto process, resulting in inefficient handling and waste. For example,the iceberg lettuce head cannot be used when separating individualleaves to allow for cleaning and the production of individual lettuceleaves. The deeply cup-shaped leaves make thorough cleaning difficultand this same shape prevents their use as individual leaves insandwiches because of their inability to lie flat and thus they arebroken and damaged. In contrast, such disadvantages are not present inthe shape of romaine lettuces.

Another disadvantage of existing iceberg varieties lies in their growingpattern. Because they grow low to the ground, harvesting must be done byhand. This means high harvest costs due to hand labor, which can alsocause potential damage to the head when the lettuce stem is not cut atthe correct level. Improperly harvested heads cannot be salvaged andmust be discarded. In contrast, romaine lettuces grow erect and theirleaves are oriented nearly vertically, thereby allowing for betteraccess to the stem. These erect plants (such as a romaine) make theprospect of machine harvesting possible. Machine harvesting results insignificant time and money savings.

Another disadvantage of existing iceberg varieties lies in theirsusceptibility to certain debilitating diseases. All plants rely onevaporation of moisture from the surface of their leaves to draw vitalwater and nutrients up into the remote areas where growth occurs. Due tothe round head shape for crisphead lettuce, inner leaves are not exposedto light and air, thereby inhibiting evaporation of moisture from itsinner leaves and the translocation of critically needed water,nutrients, and defensive agents to all interior areas of the head. Underthese circumstances, the lack of calcium and other minerals in theprocess of leaf formation is a common problem in iceberg lettuce,causing brown and black spots (tipburn) to occur leading to thelikelihood of subsequent infection by secondary pathogens such asbacteria and fungi, that result in the rapid decay and spoilage of thehead from the inside out. In contrast, such diseases are much rarer inromaine lettuces due to the erect, semi-open shape of the head.

Other problems with existing cultivars adapted to western conditionsinclude a lack of resistance to corky root rot and lettuce mosaic virus.Corky root rot is believed to be caused by a pathogenic soil bacteriumof the genus Rhizomonas. One species of Rhizomonas that is commonlyfound to cause corky root rot is R. suberifaciens. Corky root rotaccounts for significant lettuce crop loss in the western United States,particularly in the valleys of the central coast of California, i.e.,the Salinas, Santa Maria, and Lompoc valleys.

Lettuce mosaic virus, on the other hand, is commonly found throughoutthe world, and occurs in all lettuce production areas of the UnitedStates. Vectored by the Green Peach aphid (Myzus persicae), outbreaks oflettuce mosaic virus can devastate an entire field within a short periodof time.

Corky root rot symptoms include yellow bands on tap and lateral roots oflettuce seedlings. Guide to Leafy Vegetable Production in the Far West,Ron Smith, ed., California-Arizona Farm Press (1997). Yellow areasgradually expand and develop a green-brown color with cracks and roughareas on the root surface. The entire taproot may become brown, severelycracked and may cease to function. Feeder root systems are reduced anddamaged. Roots become very brittle and break off easily. When the rootis severely discolored, aboveground symptoms show up as wilting duringwarm temperatures, stunting and general poor, uneven growth. Loss of theroot system results in stunted plants that are chlorotic and too smallto harvest.

Lettuce mosaic virus symptoms first appear as vein clearing in the newlydeveloped leaves followed by mottling (a mosaic appearance) andrecurving of the leaves as they mature. Ryder, E. J., Leafy SaladVegetables, AVI Publishing Company. The leaf margins increase inundulation and necrotic spots may also appear. Infected plants aregenerally much smaller than healthy plants, rendering the plantsunsuitable for harvest.

SUMMARY OF THE INVENTION

The present invention relates to a new type of lettuce which combines anumber of advantageous characteristics of romaine, e.g., a flat leafshape, stature and semi-open headedness plus iceberg lettucecharacteristics, e.g., large, spherical, firm heads with inner blanchedleaves and a crunchy, bland texture.

The present invention relates to an iceberg lettuce plant having a firstouter leaf having a length to width ratio of between about 1.2 and about2.7. The present invention further relates to iceberg lettuce plantshaving spatulate leaf shape or an elliptical stature or a semi-open headformation. This invention is also directed to methods of producing aniceberg lettuce with a first outer leaf having a length to width ratioof between about 1.2 and about 2.7.

DETAILED DESCRIPTION OF THE INVENTION

Definitions—In the description and tables which follow, a number ofterms are used. In order to provide a clear and consistent understandingof the specification and claims, including the scope to be given suchterms, the following definitions are provided:

First outer leaf—As described herein, “first outer leaf” means the firstleaf located on the outer surface of the lettuce head.

Leaf Length to Width Ratio (L/W Ratio)—As used herein, the length towidth ratio is calculated by dividing the length of the first outer leafby the width of the first outer leaf measured at the widest point.

Outer leaf—As used herein, the term “outer leaf” refers to the 10outermost leaves on a head of lettuce.

Inner leaf—As used herein, the term “inner leaf” refers to the 10 leavesclosest to the core of a head of lettuce.

This invention provides a new type of iceberg lettuce having a plant andleaf shape and stature similar to romaine lettuce. At maturity, theheads of the plants of the present invention generally measure more than20.0 cm in length and are similar to a typical romaine plant height,whereas normal iceberg heads rarely exceed 20.0 cm.

A common problem in the creation of new varieties is the very lowoccurrence of the desired genetic phenotype in a large geneticpopulation. With more than 10,000 genes known to exist in plants, it isoften highly improbable, if not impossible to converge on all thedesired genetic traits in one individual. Prior to this work, it was notknown whether the modified shape and stature of romaine could beincorporated into a commercially viable iceberg lettuce variety.Previous work with iceberg x romaine combinations was not able to detectthe unique genetic concurrence of stature and internal quality of thepresent invention.

Another common problem that may occur includes gene interactions,whereby the products of the genes interact. Such interaction may preventthe expression of a selected desirable trait. One form of geneinteraction is epistasis, whereby interaction between products ofnon-allelic genes results in modification or masking of the desiredphenotype. Epistasis may be brought about by modification of genefunction due to alterations in the signal-transducing pathway. Epistasismay also occur indirectly through non-intracellular phenomena. Forexample, a plant with certain metabolites may exert deleterious effectson a developing embryo which does not normally produce such metabolites.

Another problem in the creation of new varieties includes linkage drag,whereby undesirable genes are brought into a population on the basis ofhitchhiking if no recombination occurs between the selected desirableand undesirable genes.

In one aspect of the invention, methods for developing novel plant typesare presented. In one preferred embodiment the specific type of breedingmethod is pedigree selection, where both single plant selection and massselection practices are employed. Pedigree selection, also known as the“Vilmorin system of selection,” is described in Fehr, Walter; Principlesof Cultivar Development, Volume I, Macmillan Publishing Co., which ishereby incorporated by reference.

In general, selection is first practiced among F₂ plants. In the nextseason, the most desirable F₃ lines are first identified, then desirableF₃ plants within each line are selected. The following season and in allsubsequent generations of inbreeding, the most desirable families areidentified first, then desirable lines within the selected families arechosen, and finally desirable plants within selected lines are harvestedindividually. A family refers to lines that were derived from plantsselected from the same progeny row the preceding generation.

Using this pedigree method, two parents may be crossed using anemasculated female and a pollen donor (male) to produce F₁ offspring. Tooptimize crossing, it is important to note that lettuce is an obligateself-pollinating species. This means that the pollen is shed beforestigma emergence, assuring 100% self-fertilization. Since each lettuceflower is an aggregate of about 10-20 individual florets, manual removalof the anther tubes containing the pollen is tedious. As such, a methodof misting to wash the pollen off prior to fertilization may be employedto assure crossing or hybridization.

Any varieties of iceberg or romaine lettuce may be used as parents inthe method of the present invention. More preferably, the parentalvarieties are selected from commercial varieties that individuallyexhibit one or more desired phenotypes. Also any breeding methodinvolving selection of plants for the desired phenotype can be used inthe method of the present invention.

The F₁ may be self-pollinated to produce a segregating F₂ generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F₃, F₄, F₅, etc.) until the traits arehomozygous or fixed within a breeding population.

Although a number of different selection criteria may be used, theselection criteria may include one or more of the following:

1. identification of an iceberg cultivar with the shape and stature of aromaine lettuce

2. a semi-open head

3. dark outer color with blanched inner color

4. resistance to corky root rot

5. resistance to lettuce mosaic virus

6. length to width ratio of greater than 1.0, and

7. plant height.

The pathogen responsible for corky root is Rhizomas suberifaciens. CA1is the most common strain and is publicly available from the ATCC(Accession No. 49355). Other useful strains include CA3 and CA15.Colonies of R. suberifaciens are initially translucent but later becomeopaque. The colonies are umbonate, compact colonies, which ultimatelybecome wrinkled and have raised edges on S-medium as described in VanBruggen, et al. 1990, Host Range of Rhizomas suberifaciens, the causalagent of corky root of lettuce, Plant Disease, 74:581-584.

R. suberifaciens is an aerobic bacterium, ranging in morphology fromsmall (0.6-1.4 mu by 0.3-0.6 mu) rods with one lateral flagellum to longfilaments. According to the KOH stringiness test, the bacteria seemedgram-positive, but with Hucker's gram-stain the bacteria stain wasgram-negative.

The type CA1 and other equivalent strains are publicly available in theSalinas Valley of California growing in the soil of the lettuce fields.These strains are quite common and can be isolated using the baitingprocedure described in Example 5. Characterized strains are alsoavailable from Dr. Ariena Van Bruggen at the University of California atDavis.

An initial screen for corky root is initiated using the screeningprocedure of Example 5. The seeds are sown under greenhouse-controlledconditions in vermiculite soil with a heavy concentration (approx. 10⁷cfu/ml) of corky root bacteria. Susceptibility is determined by visuallyinspecting the tap roots for greenish-yellow to gold oblong lesions.Advanced symptoms include a greenish-brown to golden root system and acorky and brown taproot.

The major pathogenic race or pathotype of corky root is CA1, but otherstrains are known. The cor gene is recognized as a gene responsible forresistance.

Lettuce Mosaic Virus (LMV) is a potyvirus. Natural transmission of LMVis achieved by the green peach aphid (Myzus persicae) feeding oninfected host leaves. Wild lettuce and bristly ox-tongue are the twomost important natural hosts. The virus is non-persistent in the aphidand seed-borne. The common strain is available from the ATCC. The ATCCaccession number is PV-63. Infected leaf tissue can be convenientlystored in a freezer at −20° Celsius.

Individual plants are inoculated with LMV as described in Example 6. Thevirus in ground leaves infected with LMV is mixed with a bufferedsolution and abraded with carborundum or sand onto the leaf surfaces ofthe test plants. Inoculation with LMV is at stand establishment when theplants have recovered from transplant shock. Stand establishment isabout 3-4 weeks after transplanting, when the plants present two orthree expanded leaves. Waiting until stand establishment increases theefficiency of screening for viral-induced mosaic symptoms.

Resistance to LMV is considered under the control of a single recessivegene. Resistant varieties are publicly available as described in Example6 and can be used as starting material for introducing LMV into thepresent invention.

The development of all the lines discussed herein (PSR 4569, PSR 6425,PSR 6595, and PSR 6032) used similar selection techniques, although theparental combinations used to make each of the crosses were different.The main factor uniting these combinations of the present invention isthe unique phenotype. This invention was facilitated by the selectioncriteria used in the breeding of these lines where the combination of aromaine shape and stature was linked to other qualities of an iceberglettuce.

During the breeding and selection of the lines disclosed herein, therewas no anticipated phenotype (expected biological design) due to thelack of prior work in this area of lettuce research and breeding. Oncean acceptable phenotype had been identified, it became clear that thesame desired characteristics could be obtained from a number ofdifferent parental combinations. However, this could only be done whenthe appropriate selection criteria were applied. Thus, only by makingrepeated selections for the desired phenotype could this invention berepeatedly produced.

The leaf shape of the present invention is spatulate. The term“spatulate” as used in this patent application includes leaf shapesvarying from narrow elliptic or oblong to oblanceolate or broadobtrullate as described in TG/13/7, page 19 supra.

The stature of the present invention is elliptical. The term“elliptical” as used in this patent application includes staturesvarying from narrowly elliptical to broadly elliptical as described inTG/13/7, page 18 supra.

The length to width ratio of the outer leaf of the present invention isapproximately 1.2 to 2.1. However, other embodiments of the inventioncan comprise ratios varying from about 1.2 to about 2.7. Preferably, thelength to width ratio can vary from 1.25 to 2.2, most preferably 1.3 to2.0.

Using Royal Horticultural Society's Color Chart the outer leaf color ofthe present invention is normally green (RHS 146B). However, otherembodiments of the invention can comprise outer leaf colors varying fromdark green (RHS 146A) to green (RHS 146B).

The lettuces of the present invention may have a semi-open headformation. The term “semi-open” as used in this patent applicationincludes head formations varying from medium to strong as described inTG/13/7, page 13, heading 9 supra.

Although the forgoing invention has been described and illustrated, itshould be understood that certain changes and modifications may bepracticed within the scope of this invention without departing from thescope of the invention as set forth in the accompanying claims.

EXAMPLES Example 1

Development and characteristics of lettuce cultivar PSR 4569

PSR 4569 originated in 1993 with the cross of the romaine PI 206964 bycultivar Salinas 88; PI 206964 was obtained from the USDA's WesternRegional Plant Introduction Station, Pullman, WA and Salinas 88 is apublic variety released by the USDA in 1978. Single plant selectionswere made in subsequent years in the area of intended commercialization.By F₆, a group of 15 families was judged uniform and bulked for trialingand seed increase.

In 1993, the romaine variety PI 206964 which is an exotic plantintroduction (PI), was crossed as the female parent with the crispheadlettuce cultivar Salinas 88 as the male parent using traditional crosshybridization techniques. PI 206964 is a romaine lettuce known to be asource of resistance to corky root rot and was obtained from the USDA'sWestern Regional Plant Introduction Station, Pullman, Wash. Salinas 88is a public variety released by the USDA in 1978; it is a crispheadlettuce known to be a source of resistance to lettuce mosaic virus andwas obtained from the United States Department of Agriculture, ARS, 1636East Alisal Street, Salinas, CA 93905. The cross between the exoticromaine parent PI 206964 and Salinas 88 resulted in an unexpected newcombination of characteristics, wherein an iceberg lettuce type had ataller plant stature, a larger length to width ratio plus other romainelettuce characteristics.

This new combination of traits was derived from the breeding andselection techniques of the present invention. Using these techniques, aplant breeder can use any iceberg lettuce and any romaine lettuce withthe method of the present invention to develop new varieties of iceberglettuce having a first outer leaf with a length to width ratio of morethan 1.0 and other romaine characteristics.

In 1994, the resulting F₁ seed from the cross was collected, planted,and allowed to self-pollinate. The resulting F₂ seed was collected. In1995, the F₂ seed was planted. From the resulting plants, five plantswere selected. The selection criteria for these plants was an iceberglettuce with the outer appearance of a romaine (e.g., spatulate leafshape, elongated, elliptical stature), but with all othercharacteristics of an iceberg (e.g., color, texture, taste and density),a dark outer color with blanched inner color, with resistance to corkyroot rot (from the PI 206964 parent), and resistance to lettuce mosaicvirus (from the Salinas 88 parent). Those five plants were allowed toself-pollinate and the resulting F₃ seed collected. In 1996, the F₃ seedwas divided into two lots. One lot was planted in a field located nearthe city of Santa Maria in Central California (PSR 4569) and the otherin a field located near Yuma, Arizona (PSR 4570, described below). Individing the seed into two lots, the aim was to breed progeny from thesame cross in two distinct climatic zones to select plants adapted tothe two major lettuce production areas in California, summer productionin Central California and winter production in Southern California andArizona. From the resulting plants, eight single plants were selectedusing the same selection criteria employed for the selection of the F₂plants. Those plants were allowed to self-pollinate and the resulting F₄seed collected. In 1997, the F₄ seed was planted in a field located nearthe city of Santa Maria in Central California. From the resultingplants, 21 plants were selected using the same selection criteriaemployed for the selection of the F₂ and F₃ plants. The 21 plants werethen self-pollinated and the resulting F₅ seed was collected. In 1998,the F₅ seed was planted in a field located near Santa Maria, in CentralCalifornia. From the resulting plants, 25 plants were selected using thesame selection criteria employed for the selection of the F₂, F₃, and F₄plants. The 25 plants were then self-pollinated and the resulting F₆seed was collected. In 1999, the F₆ seed was planted in a field locatednear Salinas, Central California. From the resulting plants, 44 highlyuniform plants were selected using the same selection criteria employedfor the selection of the F₂, F₃, F₄, and F₅ plants. The number PSR 4569was assigned and the 44 plants were then self-pollinated and theresulting F₇ seed was collected. In 2000, the F₇ seed was bulked andtrialed in growers' fields in Central California. During trials of PSR4569 in 1999 and 2000, neither genetic variants nor off-types have beenobserved in more than 9,000 plants. This was an experimental trial inorder to evaluate PSR 4569 in field conditions.

PSR 4569 has unique characteristics which are ideally suited for theAmerican fresh vegetable industry. PSR 4569 has head height similar toromaine and head diameter narrower than both romaine and iceberg, due tothe compact nature of its head (a crisphead characteristic) butelongated to a romaine height (Table 1). Its leaf color, internalqualities, and nutritional components all come from its crispheadbackground. Its exterior and interior color, head weight, leafthickness, and tendency to flowering (number of days to 15 cm.), as wellas the total composition of vitamins and minerals are all typical ofcrisphead. Such internal qualities contribute to the crisphead type,e.g. crisp interior leaves (leaf thickness), pale exterior and interiorcolor, and a distinct iceberg taste (nutritional components).Potentially mistaken for a romaine cultivar, PSR 4569 is clearly acrisphead in all aspects except for its shape.

PSR 4569 is adapted to all coastal production areas of CentralCalifornia. It contains the mo gene conferring resistance to lettucemosaic virus (the mo gene was released by the USDA in 1975) and the corgene, conferring resistance to corky root rot, strain CA1 (the cor genewas released by the Wisconsin State Experiment Station in 1978). Due tothe size and shape of this cultivar, the breeding strategy requiredcareful study and selection of this phenotype, without which thediscovery of this invention would not have been possible.

PSR 4569 is a vigorous cultivar that has a healthy root system. Thehealthy root system is a result of the cultivar's resistance to corkyroot rot. All PSR 4569 plants tested for resistance to corky root rothad scores of 0 (resistant, based on rating system of 0-5, Dr. A. H. C.van Bruggen, U.C. Davis, California Lettuce Research Program, AnnualReport, 1987, pp: 30-41) and were clean of any lesions on the rootscaused by bacterial infection. The susceptible control, cultivar GreenTowers, all had scores of 4 or 5 (susceptible) and showed lesions ontheir roots. More specifically, PSR 4569 has a root system with highroot mass. Typically, iceberg cultivars that are susceptible to corkyroot rot have a root system that has a low root mass. Because of thislow root mass, such cultivars require frequent watering, specifically, 3to 4 irrigations after thinning, in order to prevent them from dryingout and having a small head size.

TABLE 1 Lactuca sativa cultivar PSR 4569 has the following morphologicaland other characteristics: Seed Color: Black Light Dormancy: Notrequired Heat Dormancy: Susceptible Shape of Cotyledons: Spatulate LeafShape: Spatulate Leaf Length/Width Leaf Ratio: 1.5 Leaf Margin IncisionDepth: Moderate Leaf Margin Indentation: Shallowly Dentate Leaf ApicalMargin Undulation: Moderate Leaf Color: Dark Green, RHS 146A LeafAnthocyanin Distribution: Absent Leaf Glossiness: Moderate LeafBlistering: Absent Head Formation: Semi-open Leaf Thickness: Thick, 1.1± 0.2 mm Plant Height: Tall, 27.5 ± 0.9 cm Head Diameter: Medium, 17.2 ±2.4 cm Head Weight: Heavy, 1328 ± 9.8 g Tendency to Bolt: Moderate, 66days to 15 cm

PSR 4569 is the subject of a PVP application in the United States(#200000266).

Example 2

Development and characteristics of lettuce breeding line PSR 4570

In 1996, the second lot of F₃ seed described in Example 1 was planted ina field located near Yuma, Ariz. From the resulting plants, six singleplants were selected using the same selection criteria employed for theselection of the F₂ plants. Those plants were allowed to self-pollinateand the resulting F₄ seed collected. In 1997, the F₄ seed was planted ina field located near Yuma, Ariz. From the resulting plants, 16 plantswere selected using the same selection criteria employed for theselection of the F₂ and F₃ plants. The 16 plants were thenself-pollinated and the resulting F₅ seed was collected. In 1998, the F₅seed was planted in a field located near Yuma, Ariz. From the resultingplants, 17 plants were selected using the same selection criteriaemployed for the selection of the F₂, F₃, and F₄ plants. The 17 plantswere then self-pollinated and the resulting F₆ seed was collected. In1999, the F₆ seed was planted in a field located near Yuma, Ariz. Fromthe resulting plants, 34 highly uniform plants were selected using thesame selection criteria employed for the selection of the F₂, F₃, F₄,and F₅ plants. The number PSR 4570 was assigned and the 34 plants werethen self-pollinated and the resulting F₇ seed was collected. In 2000,the F₇ seed was bulked and trialed in growers' fields. During trials ofPSR 4570 in 1999 and 2000, neither genetic variants nor off-types havebeen observed in more than 10,000 plants. This was an experimental trialin order to evaluate PSR 4570 in field conditions.

Like PSR 4569, PSR 4570 has unique characteristics which are ideallysuited for the American fresh vegetable industry. PSR 4570 is similar toPSR 4569 except that PSR 4569 was selected for cultivation in productionareas of Coastal California, while PSR 4570 was selected for cultivationin production areas of Southern California and Arizona. The resultingdifferences are two-fold. PSR 4570 is more vigorous than PSR 4569.Harvest weights for PSR 4570 average 10% higher than PSR 4569. Also, theoverall size of PSR 4570 is larger and plant diameter wider than PSR4569. The other difference is in the extent of leaf undulation. PSR 4569has a higher degree of leaf undulation than does PSR 4570.

PSR 4570 is adapted to desert production areas of Southern Californiaand Arizona. It contains the cor gene, conferring resistance to corkyroot rot, strain CA1 (the cor gene was released by the Wisconsin StateExperiment Station in 1982), but does not contain the mo gene,conferring resistance to lettuce mosaic virus. Due to the size and shapeof this cultivar, the breeding strategy required careful study andselection of this phenotype, without which the discovery of thisinvention would not have been possible.

PSR 4570 is a vigorous cultivar that has a healthy root system. Thehealthy root system is a result of the cultivar's resistance to corkyroot rot. All PSR 4570 plants tested for resistance to corky root rothad scores of 0 (resistant, using the same rating system as with PSR4569) and were clean of any lesions on the roots caused by bacterialinfection. The susceptible control, cv. Green Towers, all had scores of4 or 5 (susceptible) and showed lesions on their roots. Morespecifically, PSR 4570 has a root system with high root mass. Typically,iceberg cultivars that are susceptible to corky root rot have a rootsystem that has a low root mass. Because of this low root mass, suchcultivars require frequent watering, specifically, 3 to 4 irrigationsafter thinning, in order to prevent them from drying out and having asmall head size.

TABLE 2 Lactuca sativa cultivar PSR 4570 has the following morphologicaland other characteristics: Seed Color: Black Light Dormancy: Notrequired Heat Dormancy: Susceptible Shape of Cotyledons: Spatulate LeafShape: Spatulate Leaf Length/Width Leaf Ratio: 1.5 Leaf Margin IncisionDepth: Moderate Leaf Margin Indentation: Shallowly Dentate Leaf ApicalMargin Undulation: Moderate Leaf Color: Dark Green, RHS 146A LeafAnthocyanin Distribution: Absent Leaf Glossiness: Moderate LeafBlistering: Absent Head Formation: Semi-open Leaf Thickness: Thick, 1.2± 0.2 mm Plant Height: Tall, 28.0 ± 1.2 cm Head Diameter: Medium, 20.2 ±2.5 cm Head Weight: Heavy, 1439 ± 10.4 g Tendency to Bolt: Moderate, 61days to 15 cm

Example 3

Development and characteristics of lettuce breeding line PSR 6425

Breeding line PSR 6425 originated from the crosses Green Towers XBarcarole and PI 289059A X PSR 11093 in 1991. Green Towers was releasedby Harris Moran Co. in 1988, Barcarole is an old European romainevariety, PI289059A was obtained from the USDA's Western Regional PlantIntroduction Station, Pullman, Wash., and PSR 11093 is an internalbreeding line of Seminis Vegetable Seeds, Inc. In 1992, the resulting F₁seed from these two crosses were collected, planted, and allowed toself-pollinate. The resulting F₂ seed was collected. In 1993, the F₂seed was planted. From the resulting plants, 8 plants of the first crossand 9 plants of the second cross were selected. The selection criteriafor the first cross was a romaine lettuce with typical characteristics(e.g., spatulate leaf shape, elongated, elliptical stature, and asemi-loose arrangement of leaves arranged in a cylindrical shape), whilethe selection criteria for the second cross was an iceberg lettuce withtypical characteristics (e.g., spherical shape, pale green color, crisptexture, bland taste, and a firm density). The selected plants wereallowed to self-pollinate and the resulting F₃ seed collected. In 1994,the F₃ seed was sown from plants of both crosses. From the resultingplants, 12 plants were selected from the first cross and 14 plants wereselected from the second cross, using the same selection criteriaemployed for the selection of the F₂ plants. Those plants were allowedto self-pollinate and the resulting F₄ seed collected. In 1995, the F₄seed was planted. From the resulting plants, six plants were selectedfrom the first cross and 11 plants from the second cross using the sameselection criteria employed for the selection of the F₂ and F₃ plants.Those plants were then self-pollinated and the resulting F₅ seed wascollected for both crosses. In 1996, the F₅ seed was planted, raised tothe flowering stage, and then crossed with the first cross (Green TowersX Barcarole) F₅ used as the female parent and the second cross (PI289059A X PSR 11093) F₅ used as the male parent. From the cross, F₁ seedwas planted in 1997 and allowed to self-pollinate. The resulting F₂ seedwas collected. In 1998, the F₂ seed was planted. From the resultingplants, 10 plants were selected. The selection criteria for these plantswas an iceberg lettuce with the outer appearance of a romaine (e.g.,spatulate leaf shape, elongated, elliptical stature), but with all othercharacteristics of an iceberg (e.g., color, texture, taste and density),and a dark outer color with blanched inner color. Of the resultingplants, 20 plants were selected. The 20 plants were then self-pollinatedand the resulting F₃ seed was collected. In 1999, the F₃ seed wasplanted. From the resulting plants, 15 plants were selected using thesame selection criteria employed for the selection of the F₂ plants. In2000, F₄ plants were selected using the same selection criteria employedfor the selection of the F₂ and F₃ plants.

TABLE 3 Lactuca sativa cultivar PSR 6425 has the following morphologicaland other characteristics: Seed Color: White Light Dormancy: Notrequired Heat Dormancy: Susceptible Shape of Cotyledons: Spatulate LeafShape: Spatulate Leaf Length/Width Leaf Ratio: 1.3 Leaf Margin IncisionDepth: Mostly Entire Leaf Margin Indentation: Slightly Dentate LeafApical Margin Undulation: Moderate Leaf Color: Dark Green, RHS 146A LeafAnthocyanin Distribution: Absent Leaf Glossiness: Moderate LeafBlistering: Absent Leaf Thickness: Thick, 1.0 ± 0.3 mm Plant Height:Medium, 20.0 ± 1.6 cm Head Diameter: Medium, 14.0 ± 1.3 cm HeadFormation: Semi-open Head Weight: Medium, 926 ± 6.9 g Tendency to Bolt:Moderate, 61 days to 15 cm

Due to the phenotype of this cultivar, the breeding methods requiredspecific selection of this specialized plant shape, without which thediscovery of this invention would not have been possible. PSR 6425 issimilar to PSR 4569 in its overall shape and appearance, however it hasa smaller head (60% as tall and 50% as heavy as PSR 4569), and its headis more exposed on top. Leaf margins are nearly smooth, compared to aslight undulation for PSR 4569, while leaf color is similar. Like PSR4569, PSR 6425 is adapted to the production areas of California andArizona.

Example 4

Development and characteristics of lettuce breeding line PSR 6595

Breeding line PSR 6595 originated from the crosses Augustus X El Doradoand Moraleja X Clemente in 1991. Cultivars Augustus, El Dorado,Moraleja, and Clemente are all varieties sold by Seminis VegetableSeeds, Inc. In 1992, the resulting F₁ seed from these two crosses werecollected, planted, and allowed to self-pollinate. The resulting F₂ seedwas collected. In 1993, the F₂ seed was planted. From the resultingplants, three plants of the first cross and 11 plants of the secondcross were selected. The selection criteria for the first cross were aniceberg lettuce with typical characteristics (e.g., spherical shape,pale green color, crisp texture, bland taste, and a firm density), whilethe selection criteria for the second cross was a romaine lettuce withtypical characteristics (e.g., spatulate leaf shape, elongated,elliptical stature, and a semi-loose arrangement of leaves arranged in acylindrical shape). The selected plants were allowed to self-pollinateand the resulting F₃ seed collected. In 1994, the F₃ seed was sown fromplants of both crosses, raised to the flowering stage, and then crossedwith the first cross (Augustus X El Dorado) F₃ used as the female parentand the second cross (Moraleja X Clemente) F₃ used as the male parent.From the cross, F₁ seed was planted in 1995 and allowed toself-pollinate. The resulting F₂ seed was collected. In 1996, the F₂seed was planted. From the resulting plants, seven plants were selected.The selection criteria for these plants was an iceberg lettuce with theouter appearance of a romaine (e.g., spatulate leaf shape, elongated,elliptical stature), but with all other characteristics of an iceberg(e.g., color, texture, taste and density), and a dark outer color withblanched inner color. Of the resulting plants, 10 plants were selected.The 10 plants were then self-pollinated and the resulting F₃ seed wascollected. In 1997, the F₃ seed was planted. From the resulting plants,six plants were selected using the same selection criteria employed forthe selection of the F₂ plants. In 1998 F₄ plants were selected usingthe same selection criteria employed for the selection of the F₂ and F₃plants. From the resulting plants, 16 plants were selected andself-pollinated and the resulting F₅ seed was sown in 1999. Of theresulting plants, 10 plants were selected and allowed to self-pollinateresulting in F₆ seed.

TABLE 4 Lactuca sativa cultivar PSR 6595 has the following morphologicaland other characteristics: Seed Color: White Light Dormancy: Notrequired Heat Dormancy: Susceptible Shape of Cotyledons: Spatulate LeafShape: Spatulate Leaf Length/Width Leaf Ratio: 2.1 Leaf Margin IncisionDepth: Deep Leaf Margin Indentation: Coarsely Dentate Leaf Apical MarginUndulation: Slight Leaf Color: Dark Green, RHS 146A Leaf AnthocyaninDistribution: Absent Leaf Glossiness: Moderate Leaf Blistering: SlightHead Formation: Semi-open Leaf Thickness: Medium, 0.9 ± 0.2 mm PlantHeight: Medium, 19.6 ± 0.8 cm Head Diameter: Medium, 14.1 ± 2.3 cm HeadWeight: Medium, 914 ± 9.9 g Tendency to Bolt: Moderate, 61 days to 15 cm

Because of the special phenotype, the breeding needed to select the sizeand shape were specific to this cultivar. This unique plant design wouldnot have been noticed, if the detailed selection criteria were notapplied. PSR 6595 is also similar to PSR 4569 in its overall shape andappearance, however it has a less compact head and a looser arrangementof leaves around its head. PSR 6595 is 80% as tall and 60% as heavy asPSR 4569, and the head is also more exposed on top. Leaf margins aresimilar to PSR 4569, but leaf color is darker. Like PSR 4569, PSR 6595is adapted to the production areas of California and Arizona. Itsdisease resistance levels are unknown at this time.

Example 5

Development and characteristics of lettuce breeding line PSR 6032

In 1995 the leaf lettuce variety PSR 0156 was crossed as the femaleparent with the crisphead lettuce cultivar Tiber as the male parentusing traditional cross hybridization techniques. PSR 0156 is a leaflettuce known to be a source of resistance to lettuce mosaic virusresistance and is an internal breeding line of Seminis Vegetable Seeds,Inc. Cultivar Tiber is a public variety released by the USDA in 1995; itis a crisphead lettuce known to be a source of resistance to lettucemosaic virus and was obtained from the United States Department ofAgriculture, ARS, 1636 East Alisal Street, Salinas, Calif. 93905. In1996, the resulting F₁ seed from the cross was collected, planted, andallowed to self-pollinate. The resulting F₂ seed was collected. In 1997,the F₂ seed was planted. From the resulting plants, three plants wereselected. The selection criteria for these plants was an iceberg lettucewith the outer appearance of a romaine (e.g., spatulate leaf shape,elongated, elliptical stature), but with all other characteristics of aniceberg (e.g., color, texture, taste and density), a dark outer colorwith blanched inner color, with resistance to lettuce mosaic virus (fromthe PSR 0156 and the Tiber parent). Those three plants were allowed toself-pollinate and the resulting F₃ seed collected. In 1998, the F₃ seedwas planted. From the resulting plants, five single plants were selectedusing the same selection criteria employed for the selection of the F₂plants. Those plants were allowed to self-pollinate and the resulting F₄seed collected. In 1999, the F₄ seed was planted. From the resultingplants, 11 plants were selected using the same selection criteriaemployed for the selection of the F₂ and F₃ plants. The 11 plants werethen self-pollinated and the resulting F₅ seed was collected. In 2000,the F₅ seed was planted. From the resulting plants, 10 plants wereselected using the same selection criteria employed for the selection ofthe F₂, F₃, and F₄ plants. The 10 plants were then self-pollinated andthe resulting F₆ seed was collected. In 2001, the F₆ seed was planted.From the resulting plants, 19 highly uniform plants were selected usingthe same selection criteria employed for the selection of the F₂, F₃,F₄, and F₅ plants.

TABLE 5 Lactuca sativa cultivar PSR 6032 has the following morpholicaland other characteristics: Seed Color: Black Light Dormancy: Notrequired Heat Dormancy: Susceptible Shape of Cotyledons: Spatulate LeafShape: Spatulate Leaf Length/Width Leaf Ratio: 2.0 Leaf Margin IncisionDepth: Shallow Leaf Margin Indentation: Moderately Dentate Leaf ApicalMargin Undulation: Moderate Leaf Color: Dark Green, RHS 146A LeafAnthocyanin Distribution: Absent Leaf Glossiness: Moderate LeafBlistering: Slight Head Formation: Semi-open Leaf Thickness: Medium, 0.9± 0.2 mm Plant Height: Slightly Tall, 24.8 ± 1.2 cm Head Diameter:Medium, 16.1 ± 2.5 cm Head Weight: Heavy, 1239 ± 10.1 g Tendency toBolt: Moderate, 61 days to 15 cm

Due to the size and shape of this cultivar, the breeding strategyrequired careful study and selection of this phenotype, without whichthe discovery of this invention would not have been possible. PSR 6032is also similar to PSR 4569 in its overall shape and appearance, howeverits head remains more open at maturity and its leaf margins have a muchhigher degree of undulation. PSR 6595 is as tall as PSR 4569 and 70% asheavy with a high concentration of tightly crowded leaves at the openingon top. Leaf color is also similar to that of PSR 4569, and like PSR4569, PSR 6032 is adapted to the production areas of California andArizona.

Example 6. Corky Root Inoculation Techniques

Using soil samples, the organism causing corky root can be isolated byusing 2-3 week old seedling of a susceptible variety, like “Salinas,” asbaits. Soil suspensions are made by using 50 g of soil in 75 ml ofdistilled water plus 3 drops of Tween.™ The suspensions are stirred for10-20 minutes and filtered through six layers of cheesecloth. Suspension(5 ml) is dispensed at the base of each of the five 2-3 week oldseedlings in a greenhouse. Three-four weeks after inoculation, theplants are uprooted and the bacteria is isolated from the yellow orcorked areas on the roots.

Root samples are rinsed under running tap water, sonicated in 20 ml ofsterile water, and comminuted in a sterile mortar with 10 ml of steriledistilled water. The cell suspensions obtained from the root surface bysonication and from comminuted roots are filtered through a 65 micronfilter and 0.04 ml of filtered suspension (undiluted and 10-folddiluted) is spread into plates of S-medium amended with streptomycinsulfate. The plates are incubated at 28° Celsius for 10 days. Theslow-growing colonies are identified by their translucent, later opaquecolonies in S-medium.

Bacterial cultures are stored long term at −85° Celsius. A 72-hourliquid culture in S-medium is diluted to 15% glycerol. The cultures areslightly unstable after successive transfers on solid S-medium andpathogenicity may be affected.

To increase the inoculum for screening, one cryovial is thawed and addedto 10 ml liquid S-medium. The medium is left at room temperature withcontinual shaking for 4 days. R. suberifaciens is not a vigorous growingbacteria. If growth is heavy after 2 days, then there is a stronglikelihood of contamination. The original 10 ml is used to seed largervolumes of liquid S-medium at the rate of 1 m1:1 liter. To check forcontaminants, streak 1 loop onto a plate of S-medium and CS-20. R.suberifaciens does not grow well on CS-20. After 3-4 days at roomtemperature, there will be distinct colonies on S-medium. After 5-6 daysof continual agitation/shaking at room temperature, the cultures will becloudy and turbid. The cultures are then diluted 50% with deionized,distilled water.

The diluted culture media is applied to 1020 trays of 7-day oldseedlings at a rate of at least 500 ml per tray. Any excess water in thetrays is removed prior to inoculation by sifting in the tray beforeadding inoculum.

Resistant varieties of lettuce are available and include commonlyavailable varieties such as “Greenlake, Montello, Southbay, Raleigh,Misty Day, and Glacier.” Susceptible varieties are also commonlyavailable and include “Salinas.”

The screening of lettuce seedlings conveniently takes place in K36D1cell inserts in a 1020 tray without holes. The tray combinations areavailable from Kord Products, Ltd. Bramaton, Ontario, Canada. 10-20seeds are sown per cell in vermiculite. The seedlings are kept at 17-28°Celsius throughout the test with 14 hours of light. The trays are misteddaily for at least 2 weeks and/or watered from below by adding about 1cm of water to the tray. Between waterings, the tray is allowed tobecome dry, yet the vermiculite should remain moist.

Approximately 10 days after sowing, the flats are flooded with halfstrength Hoaglands solution to supply nutrition. The seedlings arewatered every other week with 0.005M CA(NO₃)₂+0.005 KNO₃.

The seedlings are inoculated with the bacteria approximately 7-8 daysafter sowing. The preferred time is when the root system has started toexpand. The liquid in the bottom of the tray is removed and inoculum isadded to a depth of 1 cm. The seedlings are not watered for at least 24hours. The trays may be misted. A second inoculation is done 2 weekslater.

Post inoculation, the seedlings are maintained as they were during thegrowth phase. The flats are kept moist with a maximum of 1 cm of waterin the tray. The trays may be allowed to go dry; however, thevermiculite must remain wet at all times.

Susceptibility to corky root is presented by plant stunting and poorlydeveloped root systems. The tap root develops yellow to golden oblonglesions, especially where the lateral root has emerged. The entire rootsystem is brown to golden and, in advanced cases, the tap root is corkyand brown. The absence of stunting, and lesions under the conditionsprovided is evidence of resistance. The duration of test from sowing toreading is approximately 4 weeks.

Suitable techniques are described in Van Bruggen et al. 1990, Host Rangeof Rhizomas suberifaciens, the Causal Agent of Corky Root of Lettuce,Plant Disease. 74(8):581-584; Van Bruggen et al. 1990, The Effect ofCover Crops and Fertilization with Ammonium Nitrate of Corky Root ofLettuce, Plant Disease 74(8):584-589; and Van Bruggen et al. 1990,Distinction Between Infectious and Non-infectious Corky Root of Lettucein Relation to Nitrogen Fertilizer, J. Amer. Soc. Hort. Sci.115(5):762-770.

Example 7

Screening for Lettuce Mosaic Virus

Resistance to Lettuce Mosaic Virus (LMV) is determined using a standard,mechanical inoculation. Three to four weeks prior to inoculation of testplants or breeding lines, the virus is increased by inoculation onsusceptible lettuce varieties like Green Towers, Salinas or Vanguard.Resistance checks can be done using varieties Don Juan, Salinas 88 andVanguard 75. The infected leaves are then ground in a buffer solution of0.5M potassium phosphate (K2HPO₄, 8.71 g/l) at pH 7.2 in a ratio of 1 gleaf to 4 ml buffer solution. An antioxidant (mercaptoethanol) is alsoadded, as well as activated charcoal at 0.25 g. Carborundum at 0.4 g canbe added to the inoculum, then rubbed on the leaves of the test plants.

The test plants include both seedlings and mature plants. They aretransplanted in flats until they recover to their 3-4 leaf stage, whenthey are inoculated.

About 3 weeks after inoculation, plants showing symptoms are culled out,and the remaining plants grown until the flag leaf stage, when they arerated again for mottling symptoms.

Suitable references include: Pink, D. A., et al. 1992 “Differentiationof Pathotypes of Lettuce Mosaic Virus,” Plant Pathology, 41:5-12; andRyder, E. J., 1973, “Seed Transmission of Lettuce Mosaic Virus in MosaicResistant Lettuce,” J. Amer. Soc. Hort. Sci., 98(6):610-614.

DEPOSIT INFORMATION

Lettuce, Lactuca sativa L., seeds of PSR 4569 have been placed ondeposit with the American Type Culture Collection (ATCC), Manassas, Va.,under Deposit Accession Number PTA-3249 on Mar. 28, 2001.

Lettuce, Lactuca sativa L., seeds of PSR 4570 have been placed ondeposit with the American Type Culture Collection (ATCC), Manassas, Va.,under Deposit Accession Number PTA-3247 on Mar. 28, 2001.

Lettuce, Lactuca sativa L., seeds of PSR 6425; PSR 6595 and PSR 6032have been placed on deposit with the American Type Culture Collection(ATCC), Manassas, Va., under Deposit Accession Number PTA-3248 on Mar.28, 2001.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationssuch as single gene modifications and mutations, somoclonal variants,variant individuals selected from large populations of the plants of theinstant inbred and the like may be practiced within the scope of theinvention, as limited only by the scope of the appended claims.

1. An iceberg lettuce plant, and the parts thereof, comprising a firstouter leaf having a length to width ratio between about 1.2 to about2.7.
 2. An iceberg lettuce plant and the parts thereof according toclaim 1 having a spatulate leaf shape.
 3. An iceberg lettuce plant andthe parts thereof according to claim 1 having an elliptical stature. 4.An iceberg lettuce plant and the parts thereof according to claim 1,further comprising an outer leaf having a color which ranges from aboutRHS 146A to about RHS146B.
 5. An iceberg lettuce plant and the partsthereof according to claim 4, further comprising a blanched inner leafcolor, ranging from about RHS 145C to about RHS 145D.
 6. An iceberglettuce plant and the parts thereof according to claim 1, furthercomprising a semi-open head.
 7. An iceberg lettuce plant and the partsthereof according to claim 1, further comprising resistance to one ormore of the following diseases: corky root and lettuce mosaic virus. 8.A method of producing an iceberg lettuce having a first outer leaf witha length to width ratio of about 1.2 to about 2.7 comprising: a)crossing an iceberg lettuce with a romaine lettuce to produce hybridseed; b) growing said hybrid seed to produce a hybrid plant; c) selfingsaid hybrid seed to produce F₂ progeny seed; d) growing said F₂ progenyseed to produce F₂ plants; and e) selecting said F₂ plants forexpression of one or more characteristics selected from the groupconsisting of: length to width ratio greater than 1.0, spatulate leafshape, semi-open head, and elliptical stature, resistance to corky rootrot, resistance to lettuce mosaic virus, and plant height. 9-37.(canceled)